Spontaneous rearrangement of aminoalkylisothioureas into mercaptoalkylguanidines, a novel class of nitric oxide synthase inhibitors with selectivity towards the inducible isoform

Abstract

1 The generation of nitric oxide (NO) from L-arginine by NO synthases (NOS) can be inhibited by guanidines, amidines and S-alkylisothioureas. Unlike most L-arginine based inhibitors, however, some guanidines and S-alkylisothioureas, in particular aminoethylisothiourea (AETU), show selectivity towards the inducible isoform (iNOS) over the constitutive isoforms (endothelial, ecNOS and brain isoform, bNOS) and so may be of therapeutic benefit. In the present study we have investigated the effects of AETU and other aminoalkylisothioureas on the activities of iNOS, ecNOS and bNOS. 2 AETU, aminopropylisothiourea (APTU) and their derivatives containing alkyl substituents on one of the amidino nitrogens, potently inhibit nitrite formation by immunostimulated J774 macrophages (a model of iNOS activity) with EC50 values ranging from 6-30 yM (EC" values for N0-methyl-L-arginine (L-NMA) and N0-nitro-L-arginine were 159 and > 1000 yM, respectively). The inhibitory effects of these aminoalkylisothioureas (AATUs) were attentuated by L-arginine in the incubation medium, indicating that these agents may compete with L-arginine for its binding site on NOS. 3 The above AATUs undergo chemical conversion in neutral or basic solution (pH 7 or above) as indicated by (1) the disappearance of AATUs from solution as measured by h.p.l.c., (2) the generation of free thiols not previously present and (3) the isolation of species (as picrate and flavianate salts) from neutral or basic solutions of AATUs that are different from those obtained from acid solutions. 4 Mercaptoalkylguanidines (MAGs) were prepared and shown to be potent inhibitors of iNOS activity with EC50s comparable to those of their isomeric AATUs. 5 These findings suggest that certain AATUs exert their potent inhibitory effects through intramolecular rearrangement to mercaptoalkylguanidines (MAGs) at physiological pH. Those AATUs not capable of such rearrangement do not exhibit the same degree of inhibition of iNOS. 6 In contrast to their potent effects on iNOS, some AATUs and MAGs were 20-100 times weaker than NG-methyl-L-arginine and N0-nitro-L-arginine as inhibitors of ecNOS as assessed by their effects on the conversion of L-arginine to L-citrulline in homogenates of bovine endothelial cells and by their pressor effects in anaesthetized rats. Thus mercaptoalkylguanidines represent a new class of NOS inhibitors with preference towards iNOS. 7 AETU and mercaptoethylguanidine (MEG), when given as infusions, gave slight decreases in MAP in control rats. However, infusions of AETU or MEG to endotoxin-treated rats caused an increase in MAP and restored 80% of the endotoxin-induced fall in MAP.8 High doses of MEG (30-60 mg kg-') caused a decrease in MAP of normal rats. This depressor effect may be a consequence of the in vivo oxidation of MEG to the disulphide, guanidinoethyldisulphide (GED), which caused pronounced, transient hypotensive responses in anaesthetized rats and caused endothelium-independent vasodilator responses in precontracted rat aortic rings in vitro. 9 In som...

Mercaptoethylguanidine and Guanidine Inhibitors of Nitric-oxide Synthase React with Peroxynitrite and Protect against Peroxynitrite-induced Oxidative Damage

“…These observations indicate that MEG is not a direct scavenger of NO. We have previously demonstrated that MEG enhances the endothelium-dependent relaxations in response to acetylcholine (46), a finding that we were unable to explain at that point. One possibility is that MEG enhances the release of NO from SNAP.…”

“…We have observed that, in both assays, the potencies of the free thiol-containing compounds were approximately 2 orders of magnitude greater than those that cannot undergo a rearrangement to yield free thiols (46), such as S-(dimethylaminopropyl)-AETU (Table I) and S-(dimethylaminoethyl)-AETU (data not shown). Similarly, alkylation of the sulfur (S-methyl-MEG) or its oxidation to disulfide (GED) reduced the effectiveness of the compounds as scavengers of peroxynitrite.…”

“…For example, AETU may rearrange to MEG (and a small percentage to the cyclic NOS inhibitor aminothiazoline). Oxidation of MEG yields guanidinoethyl disulfide (GED), which is a potent iNOS-selective inhibitor in its own right (46,47). In this paper, we describe our findings demonstrating that MEG and related compounds are potent scavengers of peroxynitrite.…”

“…AETU and related aminoalkylisothioureas were prepared as described previously (46). MEG and mercaptopropylguanidine were prepared from AETU and APTU, respectively, as described previously (46). Peroxynitrite was synthesized as described previously (1,15,16).…”

“…SNAP was purchased from Calbiochem. AETU and related aminoalkylisothioureas were prepared as described previously (46). MEG and mercaptopropylguanidine were prepared from AETU and APTU, respectively, as described previously (46).…”